Molecules comprising a calcineurin-like binding pocket and encoded data storage medium capable of graphically displaying them

ABSTRACT

The present invention relates to crystallized molecules and molecular complexes which comprise the active site binding pocket or the FKBP12/FK506 binding pocket of calcineurin or close structural homologues to either binding pocket. This invention also relates to a data storage material encoded with the corresponding structure coordinates of those crystallized molecules or molecular complexes. Such data storage material is capable of displaying such molecules and molecular complexes as a graphical three-dimensional representation on a computer screen. In addition, this invention relates to methods of using the structure coordinates of those molecules or molecular complexes to solve the structure of homologous proteins. This invention also relates to methods of using the structure coordinates to screen and design compounds that bind to calcineurin or homologues thereof.

TECHNICAL FIELD OF INVENTION

The present invention relates to crystallized molecules and molecularcomplexes which comprise the active site binding pocket or theFKBP12/FK506 binding pocket of calcineurin or close structuralhomologues to either binding pocket. This invention also relates to adata storage medium encoded with the corresponding structure coordinatesof those crystallized molecules or molecular complexes. Such datastorage material is capable of displaying such molecules and molecularcomplexes as a graphical three-dimensional representation on a computerscreen. In addition, this invention relates to methods of using thestructure coordinates of those molecules or molecular complexes to solvethe structure of homologous proteins. This invention also relates tomethods of using the structure coordinates to screen and designcompounds that bind to calcineurin or homologues thereof.

BACKGROUND OF THE INVENTION

FK506 is an immunosuppressant that inhibits T-cell activation andproliferation [B. E. Bierer et al., Current Opinions in Immunology, 5,pp. 763-773 (1993)]. Immunosuppressants, such as FK506, are useful drugsin the treatment of transplant rejection and the prevention ofautoimmune diseases. Furthermore, such compounds are useful tools inimmune system research.

FK506 is a more recently discovered and more potent immunosuppressantthan cyclosporin. Unfortunately, FK506 is characterized by undesirablepharmacological properties, such as toxicity and poor bioavailability[P. Neuhaus et al., Lancet, 344, pp. 423-428 (1994)]. Therefore, thereremains a need for potent immunosuppressants with improvedpharmacological properties.

FK506 acts as an immunosuppressant by inhibiting T-cell signaltransduction pathways that control lymphokine transcription factors. Asa result, gene activation of various lymphokines, including IL-2, isprevented. This in turn leads to an inhibition of T-cells, andtherefore, immunosuppression.

FK506 exerts these effects in a step-wise process. Initially, FK506binds to a peptidyl prolyl isomerase, FK506 Binding Protein (“FKBP12”).This complex then binds to, and inhibits, calcineurin. Subsequent eventsinhibit signal transduction pathways, inhibit lymphokine genetranscription, and ultimately, reduce production of lymphokines, such asIL-2.

Calcineurin is a Ca²⁺-dependent serine/threonine phosphatase. It is aheterodimer composed of 2 subunits: calcineurin A (“CnA”), a 59 kDacatalytic subunit and calcineurin B (“CnB”), a 19 kDa subunit. CnAcontains a phosphatase active site and an autoinhibitory region as wellas binding sites for calmodulin and CnB. Binding of FKBP12/FK506inhibits the phosphatase activity of calcineurin against physiologicalsubstrates. FKBP12/FK506 does not, however, bind at the phosphataseactive site.

Thus, a compound may inhibit calcineurin by binding to the phosphataseactive site (“active site”), by binding to an accessory binding site,such as the FKBP12/FK506 binding site, or by binding to both sitessimultaneously. Such compounds may interact directly with calcineurinor, alternatively, may bind to FKBP12, or a FKBP12 homologue, prior tobinding to calcineurin.

FKBP12 has been characterized by its cDNA and amino acid sequences. Thecrystal structures of FKBP12, and of FKBP12 bound to FK506, have beenreported. However, this structural information has not proven useful inthe design of calcineurin inhibitors [M. V. Caffrey et al., Bioorg. Med.Chem. Lett., 21, pp. 2507-2510 (1994)].

Rat calcineurin has been characterized by its amino acid sequences andits cDNA. Human calcineurin has been characterized by its amino acidsequences and its cDNA [Guerini et al., Proc. Natl. Acad. Sci. USA, 86,pp. 9183-87 (1989)]. Knowledge of the primary structure, i.e., aminoacid sequence, of calcineurin, however, does not allow prediction of itstertiary structure. Nor does it afford an understanding of thestructural, conformational, and chemical interactions of calcineurinwith FKBP12/FK506 or other compounds or inhibitors.

The crystal structure of calcineurin has not been reported. Nor has thecrystal structure of a calcineurin homologue or a calcineurin co-complexbeen reported. The need, therefore, exists for determining the crystalstructure of calcineurin to provide a more accurate description of thestructure of calcineurin to aid in the design of improved inhibitors ofcalcineurin activity. The crystal structure of a complex comprisingcalcineurin A, calcineurin B, FKBP12, and FK506 would provide such adescription.

Calcineurin inhibitors, such as FK506, have therapeutic potential asimmunosuppressants. Specifically, such compounds may be used in thetreatment of transplant rejection and autoimmune diseases, such asrheumatoid arthritis, multiple sclerosis, juvenile diabetes, asthma,inflammatory bowel disease, and other autoimmune diseases.

SUMMARY OF THE INVENTION

Applicants have solved this problem by achieving, for the first time,the crystallization and three-dimensional structure determination of acalcineurin/FKBP12/FK506 complex and have solved the three-dimensionalstructure of that complex. This has allowed applicants to determine thekey structural features of calcineurin, particularly the shape of itsFKBP12/FK506 binding pocket and its phosphatase active site bindingpocket.

Thus, the present invention provides molecules or molecular complexesthat comprise either one or both of these binding pockets or homologuesof either binding pocket that have similar three-dimensional shapes.

The invention also provides machine readable storage medium whichcomprises the structural coordinates of either one or both of thesecalcineurin binding pockets, or similarly shaped, homologous bindingpockets. Such storage medium encoded with these data are capable ofdisplaying a three-dimensional graphical representation of a molecule ormolecular complex which comprises such binding pockets on a computerscreen or similar viewing device.

The invention also provides methods for designing, evaluating andidentifying compounds which bind to the aforementioned binding pockets.Such compounds are potential inhibitors of calcineurin or itshomologues.

The invention also provides a method for determining at least a portionof the three-dimensional structure of molecules or molecular complexeswhich contain at least some structurally similar features tocalcineurin. This is achieved by using at least some of the structuralinformation obtained for the calcineurin complex.

The invention also provides a method for crystallizing acalcineurin/FKBP12/FK506 complex and related complexes by removing theC-terminal portion of calcineurin subunit A.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 lists the atomic structure coordinates for a bovine brainCnA/CnB/FKBP12/FK506 complex as derived by X-ray diffraction from acrystal of that complex. The following abbreviations are used in FIG. 1:

-   -   “Atom type” refers to the element whose coordinates are        measured. The first letter in the column defines the element.        -   “X, Y, Z” crystallographically define the atomic position of            the element measured.        -   “B” is a thermal factor that measures movement of the atom            around its atomic center.        -   “Occ” is an occupancy factor that refers to the fraction of            the molecules in which each atom occupies the position            specified by the coordinates.        -   A value of “1” indicates that each atom has the same            conformation, i.e., the same position, in all molecules of            the crystal.

FIG. 2 depicts the structure of the calcineurin A, calcineurin B,FKBP12, and FK506 co-complex as determined from x-ray crystallography.

FIG. 3 shows a diagram of a system used to carry out the instructionsencoded by the storage medium of FIGS. 4 and 5.

FIG. 4 shows a cross section of a magnetic storage medium.

FIG. 5 shows a cross section of a optically-readable data storagemedium.

DETAILED DESCRIPTION OF THE INVENTION

The following abbreviations are used throughout the application: A = Ala= Alanine T = Thr = Threonine V = Val = Valine C = Cys = Cysteine L =Leu = Leucine Y = Tyr = Tyrosine I = Ile = Isoleucine N = Asn =Asparagine P = Pro = Proline Q = Gln = Glutamine F = Phe = PhenylalanineD = Asp = Aspartic Acid W = Trp = Tryptophan E = Glu = Glutamic Acid M =Met = Methionine K = Lys = Lysine G = Gly = Glycine R = Arg = Arginine S= Ser = Serine H = His = HistidineCnA = calcineurin subunit ACnB = calcineurin subunit B

Additional definitions are set forth in the specification wherenecessary.

In order that the invention described herein may be more fullyunderstood, the following detailed description is set forth.

Applicants have solved the three-dimensional structure of acalcineurin/FKBP12/FK506 complex using high resolution X-raycrystallography. Importantly, this has provided, for the first time, theinformation about the shape and structure of both the calcineurin activesite binding pocket and the auxiliary FKBP12/FK506 binding pocket.

Binding pockets are of significant utility in fields such as drugdiscovery. The association of natural ligands or substrates with thebinding pockets of their corresponding receptors or enzymes is the basisof many biological mechanisms of action. Similarly, many drugs exerttheir biological effects through association with the binding pockets ofreceptors and enzymes. An understanding of such associations will helplead to the design of drugs having more favorable associations withtheir target receptor or enzyme, and thus, improved biological effects.Therefore, this information is crucial in designing potential inhibitorsof calcineurin-like binding pockets.

The term “binding pocket”, as used herein, refers to a region of amolecule or molecular complex, that, as a result of its shape, favorablyassociates with another chemical entity or compound.

The term “calcineurin-like binding pocket” refers to a portion of amolecule whose shape is sufficiently similar to either the active sitebinding pocket or FKBP12/FK506 binding pocket as to bind common ligands.This commonality of shape is defined by a root mean square deviationfrom the structure coordinates of the backbone atoms of the amino acidsthat make up that binding pocket in calcineurin (as set forth in FIG. 1)of not more than 1.5 Å. How this calculation is obtained is describedbelow.

The “active site binding pocket” or “active site” of calcineurin refersto the site where dephosphorylation of a substrate occurs. In resolvingthe crystal structure of bovine brain calcineurin applicants havedetermined that calcineurin subunit A amino acids 90, 91, 92, 118, 120,121, 122, 150, 151, 156, 160, 199, 232, 253, 254, 256, 281, 282, 283,284, 306, 311, 312, and 317 are situated close enough to a phosphatemolecule present in the active site (within 8 Å) to interact with it. Itwill be readily apparent to those of skill in the art that the numberingof amino acids in other isoforms of calcineurin may be different thanthat isolated from bovine brain.

Each of those amino acids is defined by a set of structure coordinatesas set forth in FIG. 1. The term “structure coordinates” refers toCartesian coordinates derived from mathematical equations related to thepatterns obtained on diffraction of a monochromatic beam of X-rays bythe atoms (scattering centers) of a calcineurin complex in crystal form.The diffraction data are used to calculate an electron density map ofthe repeating unit of the crystal. The electron density maps are used toestablish the positions of the individual atoms within the unit cell ofthe crystal.

Those of skill in the art understand that a set of structure coordinatesfor an enzyme or an enzyme-complex or a portion thereof, is a relativeset of points that define a shape in three dimensions. Thus, it ispossible that an entirely different set of coordinates could define asimilar or identical shape. Moreover, slight variations caused byacceptable errors in the individual coordinates will have little, if anyeffect on overall shape. In terms of binding pockets, these acceptablevariations would not be expected to alter the nature of ligands thatcould associate with those pockets.

The term “associating with” refers to a condition of proximity between achemical entity or compound, or portions thereof, and a calcineurinmolecule or portions thereof. The association may benon-covalent—wherein the juxtaposition is energetically favored byhydrogen bonding or van der Waals or electrostatic interactions— or itmay be covalent.

The variations discussed above may be generated because of mathematicalmanipulations of the CnA/CnB/FK506/FKBP12 structure coordinates. Forexample, the structure coordinates set forth in FIG. 1 could bemanipulated by crystallographic permutations of the raw structurecoordinates, fractionalization of the raw structure coordinates, integeradditions or subtractions to sets of the raw structure coordinates,inversion of the raw structure coordinates or any combination of theabove.

Alternatively, modifications in the crystal structure due to mutations,additions and deletions of amino acids in any of the components thatmake up the crystal could also account for variations in structurecoordinates. If such variations are within an acceptable standard erroras compared to the original coordinates, the resulting three-dimensionalshape is considered to be the same. Thus, for example, a ligand thatbound to the active site binding pocket of calcineurin would also beexpected to bind to another binding pocket whose structure coordinatesdefined a shape that fell within the acceptable error. Such modifiedcomplexes or the binding pocket(s) thereof are also within the scope ofthis invention.

A third possible variant is an unrelated molecule or molecular complexwhich contains a binding pocket that has a similar shape as acalcineurin binding pocket. The binding pocket of that unrelatedmolecule would also be expected to bind ligands that are capable ofbinding to the calcineurin binding pocket.

Various computational analyses are therefore necessary to determinewhether a molecule or the binding pocket portion thereof is sufficientlysimilar to either of the two calcineurin binding pockets describedabove. Such analyses may be carried out in current softwareapplications, such as the Molecular Similarity application of QUANTA(Molecular Simulations Inc., Waltham, Mass.) version 3.3, and asdescribed in the accompanying User's Guide, Volume 3 pgs. 134-135.

The Molecular Similarity application permits comparisons betweendifferent structures, different conformations of the same structure, anddifferent parts of the same structure. The procedure used in MolecularSimilarity to compare structures is divided into four steps: 1) load thestructures to be compared; 2) define the atom equivalences in thesestructures; 3) perform a fitting operation; and 4) analyze the results.

Each structure is identified by a name. One structure is identified asthe target (i.e., the fixed structure); all remaining structures areworking structures (i.e., moving structures). Since atom equivalencywithin QUANTA is defined by user input, for the purpose of thisinvention we will define equivalent atoms as protein backbone atoms (N,Cα, C and O) for all conserved residues between the two structures beingcompared. We will also consider only rigid fitting operations.

When a rigid fitting method is used, the working structure is translatedand rotated to obtain an optimum fit with the target structure. Thefitting operation uses a least squares fitting algorithm that computesthe optimum translation and rotation to be applied to the movingstructure, such that the root mean square difference of the fit over thespecified pairs of equivalent atom is an absolute minimum. This number,given in angstroms, is reported by QUANTA.

For the purpose of this invention, any set of structure coordinates of amolecule or molecular complex or a binding pocket thereof that has aroot mean square deviation of conserved residue backbone atoms (N, Cα,C, O) of less than 1.5 Å when superimposed—using backbone atoms— on therelevant structure coordinates listed in FIG. 1 are consideredidentical. More preferably, the root mean square deviation is less than1.0 ÅA. Most preferably, the root mean square deviation is less than 0.5Å.

The term “root mean square deviation” means the square root of thearithmetic mean of the squares of the deviations from the mean. It is away to express the deviation or variation from a trend or object. Forpurposes of this invention, the “root mean square deviation” defines thevariation in the backbone of a protein from the backbone of calcineurinor a binding pocket portion thereof, as defined by the structurecoordinates of calcineurin described herein.

The term “least squares” refers to a method based on the principle thatthe best estimate of a value is that in which the sum of the squares ofthe deviations of observed values is a minimum.

Therefore, according to one embodiment, the present invention provides acrystallized molecule or molecular complex comprising a binding pocketdefined by structure coordinates of CnA amino acids 90, 91, 92, 118,120, 121, 122, 150, 151, 156, 160, 199, 232, 253, 254, 256, 281, 282,283, 284, 306, 311, 312, and 317 according to FIG. 1, or a homologue ofsaid molecule or molecular complex comprising a binding pocket that hasa root mean square deviation from the backbone atoms of said amino acidsof not more than 1.5 Å.

Preferably, the crystallized molecule or molecular complex comprises abinding pocket that is defined by structure coordinates of those CnAamino acids that are situated within 5 Å of the phosphate molecule inthe crystal, i.e., amino acids 90, 91, 92, 118, 120, 121, 122, 150, 151,156, 160, 281, 282, 283, 306, 311, 199, 232, and 254 according to FIG.1, or a binding pocket, wherein that has a root mean square deviationfrom the backbone atoms of those amino acids of not more than 1.5 Å.

Applicants' elucidation of the calcineurin/FKBP12/FK506 crystalstructure has also revealed the details about the FKBP12/FK506 bindingpocket. An FKBP12/FK506 complex is capable of binding to calcineurin ata site separate from the active site. Because the binding of thatcomplex inhibits calcineurin-mediated activities, the elucidation of thebinding site provides a second area on which new inhibitors may bemodelled. The crystal structure revealed a subset of calcineurin aminoacids that were close enough to interact with the bound FKBP12/FK506complex.

Therefore, according to an alternate embodiment, the invention providesa crystallized molecule or molecular complex comprising a binding pocketdefined by structure coordinates of CnA 122, 124, 159, 160, 310, 312,313, 314, 339, 341, 343, 344, 345, 347, 351, 352, 353, 354, 355, 356,359, 360, and 363, and CnB amino acids 49, 50, 114, 115, 118, 119, 121,122, 123, 124, 157, 158, 159, 161, and 162 amino acids according to FIG.1, or a homologue of said molecule or molecular complex comprising abinding pocket that has a root mean square deviation from the backboneatoms of said amino acids of not more than 1.5 Å.

According to a more preferred embodiment, the molecule or molecularcomplex comprises two binding pockets. One is defined by the structurecoordinates of the amino acids that make up the calcineurin active sitebinding pocket ± a root mean square deviation from the backbone atoms ofthose amino acids of not more than 1.5 Å. The other is defined by thestructure coordinates of the amino acids that make up the calcineurinFKBP12/FK506 binding pocket ± a root mean square deviation from thebackbone atoms of those amino acids of not more than 1.5 Å.

Even more preferred are molecules or molecular complexes that aredefined by the entire set of structure coordinates in FIG. 1 ± a rootmean square deviation from the backbone atoms of those amino acids ofnot more than 1.5 Å. An alternative more preferred embodiment of thisinvention is a molecular complex that comprises amino acids 17-392 ofCnA, amino acids 5-169 of CnB, FKBP12 and FK506.

In order to use the structure coordinates generated for theCnA/CnB/FKBP12/FK506 complex or one of its binding pockets or homologuesthereof, it is necessary to convert them into a three-dimensional shape.This is achieved through the use of commercially available software thatis capable of generating three-dimensional graphical representations ofmolecules or portions thereof from a set of structure coordinates.

Therefore, according to another embodiment of this invention is provideda machine-readable storage medium comprising a data storage materialencoded with machine readable data which, when using a machineprogrammed with instructions for using said data, is capable ofdisplaying a graphical three-dimensional representation of any of themolecule or molecular complexes of this invention that have beendescribed above.

According to one embodiment, the machine-readable storage medium iscapable of displaying a graphical three-dimensional representation of amolecule or molecular complex comprising a binding pocket defined bystructure coordinates of CnA amino acids 90, 91, 92, 118, 120, 121, 122,150, 151, 156, 160, 199, 232, 253, 254, 256, 281, 282, 283, 284, 306,311, 312, and 317 according to FIG. 1, or a homologue of said moleculeor molecular complex, wherein said homologue comprises a binding pocketthat has a root mean square deviation from the backbone atoms of saidamino acids of not more than 1.5 Å. More preferably, the binding pocketis defined by structure coordinates of CnA amino acids 90, 91, 92, 118,120, 121, 122, 150, 151, 156, 160, 199, 281, 282, 283, 306, 311, 232 and254, according to FIG. 1 ± a root mean square deviation from thebackbone atoms of said amino acids of not more than 1.5 Å.

According to another embodiment, the machine-readable storage medium iscapable of displaying a graphical three-dimensional representation of amolecule or molecular complex comprising a binding pocket defined bystructure coordinates of CnA amino acids 122, 124, 159, 160, 310, 312,313, 314, 339, 341, 343, 344, 345, 347, 351, 352, 353, 354, 355, 356,359, 360, and 363; and CnB amino acids 49, 50, 114, 115, 118, 119, 121,122, 123, 124, 157, 158, 159, 161, and 162 or a homologue of saidmolecule or molecular complex, wherein said homologue comprises abinding pocket that has a root mean square deviation from the backboneatoms of said amino acids of not more than 1.5 Å.

More preferably, the computer-readable storage medium is capable ofdisplaying a graphical three-dimensional representation of a molecule ormolecular complex that comprises two binding pockets. One binding pocketis defined by the structure coordinates of the amino acids that make upthe calcineurin active site binding pocket ± a root mean squaredeviation from the backbone atoms of those amino acids of not more than1.5 Å. The other is defined by the structure coordinates of the aminoacids that make up the calcineurin FKBP12/FK506 binding pocket ± a rootmean square deviation from the backbone atoms of those amino acids ofnot more than 1.5 Å.

Even more preferred is a machine-readable data storage medium that iscapable of displaying a graphical three-dimensional representation of amolecule or molecular complex that is defined by the structurecoordinates of all of the amino acids in FIG. 1 ± a root mean squaredeviation from the backbone atoms of those amino acids of not more than1.5 Å.

According to an alternate embodiment, the machine-readable data storagemedium comprises a data storage material encoded with a first set ofmachine readable data which comprises the Fourier transform of thestructural coordinates set forth in FIG. 1, and which, when using amachine programmed with instructions for using said data, can becombined with a second set of machine readable data comprising the X-raydiffraction pattern of a molecule or molecular complex to determine atleast a portion of the structure coordinates corresponding to the secondset of machine readable data.

FIG. 3 demonstrates one version of these embodiments. System 10 includesa computer 11 comprising a central processing unit (“CPU”) 20, a workingmemory 22 which may be, e.g, RAM (random-access memory) or “core”memory, mass storage memory 24 (such as one or more disk drives orCD-ROM drives), one or more cathode-ray tube (“CRT”) display terminals26, one or more keyboards 28, one or more input lines 30, and one ormore output lines 40, all of which are interconnected by a conventionalbidirectional system bus 50.

Input hardware 36, coupled to computer 11 by input lines 30, may beimplemented in a variety of ways. Machine-readable data of thisinvention may be inputted via the use of a modem or modems 32 connectedby a telephone line or dedicated data line 34. Alternatively oradditionally, the input hardware 36 may comprise CD-ROM drives or diskdrives 24. In conjunction with display terminal 26, keyboard 28 may alsobe used as an input device.

Output hardware 46, coupled to computer 11 by output lines 40, maysimilarly be implemented by conventional devices. By way of example,output hardware 46 may include CRT display terminal 26 for displaying agraphical representation of a binding pocket of this invention using aprogram such as QUANTA as described herein. Output hardware might alsoinclude a printer 42, so that hard copy output may be produced, or adisk drive 24, to store system output for later use.

In operation, CPU 20 coordinates the use of the various input and outputdevices 36, 46, coordinates data accesses from mass storage 24 andaccesses to and from working memory 22, and determines the sequence ofdata processing steps. A number of programs may be used to process themachine-readable data of this invention. Such programs are discussed inreference to the computational methods of drug discovery as describedherein. Specific references to components of the hardware system 10 areincluded as appropriate throughout the following description of the datastorage medium.

FIG. 4 shows a cross section of a magnetic data storage medium 100 whichcan be encoded with a machine-readable data that can be carried out by asystem such as system 10 of FIG. 3. Medium 100 can be a conventionalfloppy diskette or hard disk, having a suitable substrate 101, which maybe conventional, and a suitable coating 102, which may be conventional,on one or both sides, containing magnetic domains (not visible) whosepolarity or orientation can be altered magnetically. Medium 100 may alsohave an opening (not shown) for receiving the spindle of a disk drive orother data storage device 24.

The magnetic domains of coating 102 of medium 100 are polarized ororiented so as to encode in manner which may be conventional, machinereadable data such as that described herein, for execution by a systemsuch as system 10 of FIG. 3.

FIG. 5 shows a cross section of an optically-readable data storagemedium 110 which also can be encoded with such a machine-readable data,or set of instructions, which can be carried out by a system such assystem 10 of FIG. 3. Medium 110 can be a conventional compact disk readonly memory (CD-ROM) or a rewritable medium such as a magneto-opticaldisk which is optically readable and magneto-optically writable. Medium100 preferably has a suitable substrate 111, which may be conventional,and a suitable coating 112, which may be conventional, usually of oneside of substrate 111.

In the case of CD-ROM, as is well known, coating 112 is reflective andis impressed with a plurality of pits 113 to encode the machine-readabledata. The arrangement of pits is read by reflecting laser light off thesurface of coating 112. A protective coating 114, which preferably issubstantially transparent, is provided on top of coating 112.

In the case of a magneto-optical disk, as is well known, coating 112 hasno pits 113, but has a plurality of magnetic domains whose polarity ororientation can be changed magnetically when heated above a certaintemperature, as by a laser (not shown). The orientation of the domainscan be read by measuring the polarization of laser light reflected fromcoating 112. The arrangement of the domains encodes the data asdescribed above.

Thus, in accordance with the present invention, data capable ofdisplaying the three dimensional structure of calcineurin and portionsthereof and their structurally similar homologues is stored in amachine-readable storage medium, which is capable of displaying agraphical three-dimensional representation of the structure. Such datamay be used for a variety of purposes, such as drug discovery.

For example, the structure encoded by the data may be computationallyevaluated for its ability to associate with chemical entities. Thisprovides insight into calcineurin's ability to associate with thechemical entities. Chemical entities that are capable of associatingwith calcineurin may inhibit calcineurin. Such chemical entities arepotential drug candidates. Alternatively, the structure encoded by thedata may be displayed in a graphical format. This allows visualinspection of the structure, as well as visual inspection of thestructure's association with chemical entities.

Thus, according to another embodiment, the invention provides a methodfor evaluating the ability of a chemical entity to associate with any ofthe molecules or molecular complexes set forth above. This methodcomprises the steps of: a) employing computational means to perform afitting operation between the chemical entity and a binding pocket ofthe molecule or molecular complex; and b) analyzing the results of saidfitting operation to quantify the association between the chemicalentity and the binding pocket. The term “chemical entity”, as usedherein, refers to chemical compounds, complexes of at least two chemicalcompounds, and fragments of such compounds or complexes.

For the first time, the present invention permits the use of moleculardesign techniques to design, select and synthesize chemical entities,including inhibitory compounds, capable of binding to calcineurin-likebinding pockets. Such chemical entities may interact directly with suchpockets, in areas adjacent to those pockets or, alternatively, mayinteract with FKBP12, or a homologue or mutant of FKBP12, and theresulting complex may interact with the binding pocket. Such chemicalentities and compounds, optionally bound to FKBP12, may interact witheither or both calcineurin-like binding pockets, in whole or in part.Preferably, if the entity binds to FKBP12, the resulting complexinteracts with the calcineurin-like binding pocket that corresponds tothe FKBP12/FK506 binding site on calcineurin.

Portions of both FKBP12 and FK506 participate in the binding of thatcomplex to the FKBP12/FK506 binding site of calcineurin. Therefore,inhibitors that bind to a corresponding calcineurin-like binding sitemay be designed to mimic the interactions of that entire complex withthe binding site. Alternatively, if the inhibitor is capable ofcomplexing with FKBP12, it need only mimic the interactions of the FK506portion of the FKBP12/FK506 complex with the binding site.

The crystal structure of the FKBP12/FK506 complex has been solved andhas aided in the design of new compounds that bind to FKBP12 [D. A. Holtet al., J. Am. Chem. Soc., 115, pp. 9925-38 (1993)]. However, none ofthose compounds when bound to FKBP12 have been satisfactory ininhibiting calcineurin [M. V. Caffrey et al., Bioorg. Med. Chem. Letts.,4, pp. 2507-10 (1994)]. Accordingly, applicants' elucidation of theFKBP12/FK506 binding site on calcineurin provides the necessaryinformation for designing compounds that when bound to FKBP12 are ableto bind to the corresponding calcineurin-like binding site.

Throughout this section, discussions about the ability of an entity tobind to, associate with or inhibit a calcineurin-like binding pocketrefers to features of the entity alone, or as part of a complex withFKBP12 or naturally occurring FKBP12 isoforms and homologues. Assays todetermine if a compound binds to FKBP12 are well known in the art [M. W.Harding et al., Nature, 341, pp. 758-60 (1989); J. J. Siekierka et al.,Nature, 341, pp. 755-57 (1989)].

The design of compounds that bind to or inhibit calcineurin-like bindingpockets according to this invention generally involves consideration oftwo factors. First, the entity must be capable of physically andstructurally associating with the calcineurin-like binding pocket.Non-covalent molecular interactions important in this associationinclude hydrogen bonding, van der Waals interactions, hydrophobicinteractions and electrostatic interactions.

Second, the entity must be able to assume a conformation that allows itto associate with the calcineurin-like binding pocket directly. Althoughcertain portions of the entity will not directly participate in theseassociations, those portions of the entity may still influence theoverall conformation of the molecule. This, in turn, may have asignificant impact on potency. Such conformational requirements includethe overall three-dimensional structure and orientation of the chemicalentity in relation to all or a portion of the binding pocket, or thespacing between functional groups of an entity comprising severalchemical entities that directly interact with the calcineurin-likebinding pocket or FKBP12 or homologues thereof.

The potential inhibitory or binding effect of a chemical entity on acalcineurin-like binding pocket may be analyzed prior to its actualsynthesis and testing by the use of computer modelling techniques. Ifthe theoretical structure of the given entity suggests insufficientinteraction and association between it and the calcineurin-like bindingpocket testing of the entity is obviated. However, if computer modellingindicates a strong interaction, the molecule may then be synthesized andtested for its ability to bind to a calcineurin-like binding pocket.This may be achieved by testing the ability of the molecule to inhibitcalcineurin using the assays described in Examples 6 and 7. In thismanner, synthesis of inoperative compounds may be avoided.

A potential inhibitor of a calcineurin-like binding pocket may becomputationally evaluated and designed by means of a series of steps inwhich chemical entities or fragments are screened and selected for theirability to associate with the calcineurin-like binding pockets.

One skilled in the art may use one of several methods to screen chemicalentities or fragments for their ability to associate with acalcineurin-like binding pocket. This process may begin by visualinspection of, for example, a calcineurin-like binding pocket on thecomputer screen based on the calcineurin coordinates in FIG. 1 or othercoordinates which define a similar shape generated from themachine-readable storage medium. Selected fragments or chemical entitiesmay then be positioned in a variety of orientations, or docked, withinthat binding pocket as defined supra. Docking may be accomplished usingsoftware such as Quanta and Sybyl, followed by energy minimization andmolecular dynamics with standard molecular mechanics force fields, suchas CHARMM and AMBER.

Specialized computer programs may also assist in the process ofselecting fragments or chemical entities. These include:

-   1. GRID (P. J. Goodford, “A Computational Procedure for Determining    Energetically Favorable Binding Sites on Biologically Important    Macromolecules”, J. Med. Chem., 28, pp. 849-857 (1985)). GRID is    available from Oxford University, Oxford, UK.-   2. MCSS (A. Miranker et al., “Functionality Maps of Binding Sites: A    Multiple Copy Simultaneous Search Method.” Proteins: Structure,    Function and Genetics, 11, pp. 29-34 (1991)). MCSS is available from    Molecular Simulations, Burlington, Mass.-   3. AUTODOCK (D. S. Goodsell et al., “Automated Docking of Substrates    to Proteins by Simulated Annealing”, Proteins: Structure, Function,    and Genetics, 8, pp. 195-202 (1990)). AUTODOCK is available from    Scripps Research Institute, La Jolla, Calif.-   4. DOCK (I. D. Kuntz et al., “A Geometric Approach to    Macromolecule-Ligand Interactions”, J. Mol. Biol., 161, pp. 269-288    (1982)). DOCK is available from University of California, San    Francisco, Calif.

Once suitable chemical entities or fragments have been selected, theycan be assembled into a single compound or complex. Assembly may bepreceded by visual inspection of the relationship of the fragments toeach other on the three-dimensional image displayed on a computer screenin relation to the structure coordinates of calcineurin. This would befollowed by manual model building using software such as Quanta orSybyl.

Useful programs to aid one of skill in the art in connecting theindividual chemical entities or fragments include:

-   1. CAVEAT (P. A. Bartlett et al, “CAVEAT: A Program to Facilitate    the Structure-Derived Design of Biologically Active Molecules”. In    Molecular Recognition in Chemical and Biological Problems”, Special    Pub., Royal Chem. Soc., 78, pp. 182-196 (1989)). CAVEAT is available    from the University of California, Berkeley, Calif.-   2. 3D Database systems such as MACCS-3D (MDL Information Systems,    San Leandro, Calif.). This area is reviewed in Y. C. Martin, “3D    Database Searching in Drug Design”, J. Med. Chem., 35, pp. 2145-2154    (1992).-   3. HOOK (available from Molecular Simulations, Burlington, Mass.).

Instead of proceeding to build an inhibitor of a calcineurin-likebinding pocket in a step-wise fashion one fragment or chemical entity ata time as described above, inhibitory or other calcineurin bindingcompounds may be designed as a whole or “de novo” using either an emptybinding site or optionally including some portion(s) of a knowninhibitor(s). These methods include:

-   1. LUDI (H.-J. Bohm, “The Computer Program LUDI: A New Method for    the De Novo Design of Enzyme Inhibitors”, J. Comp. Aid. Molec.    Design, 6, pp. 61-78 (1992)). LUDI is available from Biosym    Technologies, San Diego, Calif.-   2. LEGEND (Y. Nishibata et al., Tetrahedron, 47, p. 8985 (1991)).    LEGEND is available from Molecular Simulations, Burlington, Mass.-   3. LeapFrog (available from Tripos Associates, St. Louis, Mo.).

Other molecular modelling techniques may also be employed in accordancewith this invention. See, e.g., N. C. Cohen et al., “Molecular ModelingSoftware and Methods for Medicinal Chemistry, J. Med. Chem., 33, pp.883-894 (1990). See also, M. A. Navia et al., “The Use of StructuralInformation in Drug Design”, Current Opinions in Structural Biology, 2,pp. 202-210 (1992).

Once a compound has been designed or selected by the above methods, theefficiency with which that entity may bind to a calcineurin-like bindingpocket may be tested and optimized by computational evaluation. Forexample, an effective calcineurin-like binding pocket inhibitor mustpreferably demonstrate a relatively small difference in energy betweenits bound and free states (i.e., a small deformation energy of binding).Thus, the most efficient calcineurin-like binding pocket inhibitorsshould preferably be designed with a deformation energy of binding ofnot greater than about 10 kcal/mole, preferably, not greater than 7kcal/mole. Calcineurin-like binding pocket inhibitors may interact withthe binding pocket in more than one conformation that is similar inoverall binding energy. In those cases, the deformation energy ofbinding is taken to be the difference between the energy of the freeentity and the average energy of the conformations observed when theinhibitor binds to the protein.

An entity designed or selected as binding to a calcineurin-like bindingpocket may be further computationally optimized so that in its boundstate it would preferably lack repulsive electrostatic interaction withthe target enzyme. Such non-complementary (e.g., electrostatic)interactions include repulsive charge-charge, dipole-dipole andcharge-dipole interactions. Specifically, the sum of all electrostaticinteractions between the inhibitor and the protein when the inhibitor isbound to FKBP12 or a calcineurin-like binding pocket, preferably make aneutral or favorable contribution to the enthalpy of binding.

Specific computer software is available in the art to evaluate compounddeformation energy and electrostatic interaction. Examples of programsdesigned for such uses include: Gaussian 92, revision C [M. J. Frisch,Gaussian, Inc., Pittsburgh, Pa. ©1992]; AMBER, version 4.0 [P. A.Kollman, University of California at San Francisco, ©1994];QUANTA/CHARMM [Molecular Simulations, Inc., Burlington, Mass. ©1994];and Insight II/Discover (Biosysm Technologies Inc., San Diego, Calif.©1994). These programs may be implemented, for instance, using a SiliconGraphics workstation, IRIS 4D/35 or IBM RISC/6000 workstation model 550.Other hardware systems and software packages will be known to thoseskilled in the art.

Once the calcineurin binding-pocket inhibitory entity has been optimallyselected or designed, as described above, substitutions may then be madein some of its atoms or side groups in order to improve or modify itsbinding properties. Generally, initial substitutions are conservative,i.e., the replacement group will have approximately the same size,shape, hydrophobicity and charge as the original group. It should, ofcourse, be understood that components known in the art to alterconformation should be avoided. Such substituted chemical compounds maythen be analyzed for efficiency of fit to a calcineurin-like bindingpocket by the same computer methods described in detail, above.

Another approach made possible and enabled by this invention, is thecomputational screening of small molecule data bases for chemicalentities or compounds that can bind in whole, or in part, to acalcineurin-like binding pocket. In this screening, the quality of fitof such entities to the binding site may be judged either by shapecomplementarity or by estimated interaction energy. E. C. Meng et al.,J. Comp. Chem., 13, pp. 505-524 (1992).

The structure coordinates set forth in FIG. 1 can also be used to aid inobtaining structural information about another crystallized molecule ormolecular complex. This may be achieved by any of a number of well-knowntechniques, including molecular replacement.

Therefore, in another embodiment this invention provides a method ofutilizing molecular replacement to obtain structural information about amolecule or molecular complex comprising the steps of:

a) crystallizing said molecule or molecular complex;

b) generating an X-ray diffraction pattern from said crystallizedmolecule or molecular complex; and

c) applying at least a portion of the structure coordinates set forth inFIG. 1 to the X-ray diffraction pattern to generate a three-dimensionalelectron density map of the molecule or molecular complex.

By using molecular replacement, all or part of the structure coordinatesof the CnA/CnB/FKBP12/FK506 complex as provided by this invention (andset forth in FIG. 1) can provide an accurate structure determination forall or part of an unknown crystallized molecule or molecular complexmore quickly and efficiently than attempting to determine suchinformation ab initio.

Molecular replacement provides an accurate estimation of the phases foran unknown structure. Phases are a factor in equations used to solvecrystal structures that can not be determined directly. Obtainingaccurate values for the phases, by methods other than molecularreplacement, is a time-consuming process that involves iterative cyclesof approximations and refinements and greatly hinders the solution ofcrystal structures. However, when the crystal structure of a proteincontaining at least a homologous portion has been solved, the phasesfrom the known structure provide an accurate estimate of the phases forthe unknown structure.

Thus, this method involves generating a preliminary model of a moleculeor molecular complex whose structure coordinates are unknown, byorienting and positioning the relevant portion of theCnA/CnB/FKBP12/FK506 complex according to FIG. 1 within the unit cell ofthe unknown molecule or molecular complex so as best to account for theobserved X-ray diffraction pattern of the unknown crystal. Phases canthen be calculated from this model and combined with the observed X-raydiffraction pattern amplitudes to give an approximate Fourier synthesisof the structure whose coordinates are unknown. This, in turn, can besubjected to any well-known refinement technique to provide a final,accurate structure of the unknown crystal. E. Lattman, “Use of theRotation and Translation Functions”, in Meth. Enzymol., 115, pp. 55-77(1985); M. G. Rossmann, ed., “The Molecular Replacement Method”, Int.Sci. Rev. Ser., No. 13, Gordon & Breach, New York, (1972).

The structure of any portion of any crystallized molecule or molecularcomplex that is sufficiently homologous to a portion of theCnA/CnB/FKBP12/FK506 can be resolved by this method.

In a preferred embodiment, the method of molecular replacement isutilized to obtain structural information about a molecule or molecularcomplex, wherein the complex comprises at least one catalyticallyfunctional calcineurin A subunit. The term catalytically functionalcalcineurin A subunit refers” to calcineurin A, as well as fragments andstructural homologues thereof which retain their phosphatase activity.

The structure coordinates of calcineurin as provided by this inventionare particularly useful in solving the structure of other crystal formsof the CnA/CnB/FKBP12/FK506 complex.

Furthermore, the structure coordinates of calcineurin as provided bythis invention are useful in solving the structure of calcineurinmutants, which may optionally be crystallized in co-complex with achemical entity. The crystal structures of a series of such complexesmay then be solved by molecular replacement and compared with that ofwild-type calcineurin. Potential sites for modification within thevarious binding sites of the enzyme may thus be identified. Thisinformation provides an additional tool for determining the mostefficient binding interactions, for example, increased hydrophobicinteractions, between calcineurin and a chemical entity or compound.

The structure coordinates are also particularly useful to solve thestructure of crystals of calcineurin or calcineurin homologuesco-complexed with a variety of chemical entities. This approach enablesthe determination of the optimal sites for interaction between chemicalentities, including candidate calcineurin inhibitors and calcineurin.For example, high resolution X-ray diffraction data collected fromcrystals saturated with solvent allows the determination of where eachtype of solvent molecule resides. Small molecules that bind tightly tothose sites can then be designed and synthesized and tested for theircalcineurin inhibition activity.

All of the complexes referred to above may be studied using well-knownX-ray diffraction techniques and may be refined versus 2-3 Å resolutionX-ray data to an R value of about 0.20 or less using computer software,such as X-PLOR (Yale University, ©1992, distributed by MolecularSimulations, Inc.). See, e.g., Blundell & Johnson, supra; Meth.Enzymol., vol. 114 & 115, H. W. Wyckoff et al., eds., Academic Press(1985). This information may thus be used to optimize known calcineurininhibitors, and more importantly, to design and synthesize newcalcineurin inhibitors.

In another embodiment of this invention is provided a method forpreparing a CnA/CnB/FKBP12/FK506 crystal comprising the steps of forminga molecular complex between FKBP12, FK506, calcineurin A and calcineurinB; digesting the molecular complex with a protease that removes thecalmodulin binding site and the autoinhibitory domain of calcineurin A;and crystallizing the digested complex.

In another embodiment of this invention is provided a method forpreparing a CnA/CnB/FKBP12/FK506 crystal comprising the steps of forminga molecular complex between FKBP12, FK506, calcineurin A and calcineurinB; wherein calcineurin A lacks a calmodulin binding domain and anautoinhibitory domain; and crystallizing the complex.

The autoinhibitory domain of CnA has been mapped to the C-terminal 4 kDaof that polypeptide. The calmodulin binding domain is located adjacentto the autoinhibitory domain and occupies up to 14 kDa. Thus, anN-terminal 43 kDa fragment of CnA lacks both domains [M. J. Hubbard etal., Biochemistry, 28, pp. 1868-74 (1989)]. Removal of the calmodulinbinding domain and the autoinhibitory domain does not appear to affectthe active site or FKBP12/FK506 binding sites, nor the ability of CnA tobind to CnB. Removal of the calmodulin binding site and theautoinhibitory site does, however, provide a complex that providesstable crystals, suitable for analysis by X-ray crystallography.

The removal of the autoinhibitory and calmodulin domains may be carriedout either before or after calcineurin is bound to the FKBP12/FK506complex. Removal of these domains is preferably achieved by proteolyticdigestion or through recombinant DNA techniques.

Preferably, the protease is selected from the group consisting ofclostripain, trypsin, endoproteinase Lys-C, endoproteinase Asp-N,endoproteinase Glu-C, elastase, enterokinase, restriction proteaseFactor Xa, thermolysin (Altus Biologics, Cambridge, Mass.), Il-1 betaconverting enzyme or HIV-1 protease. Most preferably, the protease isclostripain.

Preferably, the processed CnA subunit in the crystallized complex has amolecular weight of about 42 kDa.

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. It should beunderstood that these examples are for illustrative purposes only andare not to be construed as limiting this invention in any manner.

EXAMPLE 1 Purification of Calcineurin A/Calcineurin B

Bovine calcineurin was isolated from calf brains (1 year old, or less),essentially as described by Sharma and Wang. (R. K. Sharma et al., J.Biol. Chem. 261, pp. 1322-1328 (1986)). This procedure yields a mixedpopulation of calcineurin isozymes and a small proportion ofnon-calcineurin contaminants. Most of the non-calcineurin contaminantswere removed by anion exchange chromatography. The crude calcineurinfraction was exchanged into buffer A (20 mM Tris-HCl, 2 mMβ—mercaptoethanol, 1 mM magnesium acetate, 1 mM imidazole, 0.1 mM EGTA,0.1 mM PMSF, pH 7.6 at 4° C.), by dialysis or ultrafiltration, at aprotein concentration of 0.5-2 mg/ml. The protein was loaded onto acolumn (2.5×20 30 cm) of DEAE-Sepharose (Pharmacia) that had beenpre-equilibrated, at 4° C., in the same buffer. After loading theprotein, the column was washed with 2-5 column volumes of buffer A, andthen the bound proteins were eluted from the column with a lineargradient of 0-300 mM NaCl (10 column volumes), in the same buffersystem, at 4° C. The calcineurin, eluting as the main peak, was thenfractionated into numerous isoforms by hydrophobic interactionchromatography. The protein was exchanged into buffer B (50 mm Tris-HCl,1.4 M ammonium sulfate, 5% (v/v) glycerol, 2 mM β-mercaptoethanol, 1 mMEGTA, pH 7.5 at 20° C.), and loaded onto a Hydropore-HIC (Rainin) column(2.0×30 cm) pre-equilibrated in the same buffer. The calcineurinisozymes were eluted at 20° C. with a linear gradient (8, columnvolumes) from 1.4 M to 0 M ammonium sulfate, keeping the other buffercomponents constant. The first peak representing about 60% of the totalwas designated as the “major isoform.” The A and B subunits were of thismajor isoform were separated by reversed phase HPLC and SDS PAGE.Peptide mapping of the fractionated subunits, followed by amino acidsequence analysis of the peptide products, generated internal sequencedata for both subunits. When compared with independently determined cDNAsequences (and the deduced amino acid sequences) the experimentallyobtained protein sequences indicated that this major isoform consistedof intact, myristoylated calcineurin B, and intact calcineurin A. Thisprotein was concentrated by ultrafiltration and dialyzed into buffer C(25 mM Tris-HCl, 0.1 mM MnCl₂, 0.1 mM CaCl₂, 2 mM β-mercaptoethanol, pH8.0 at 4° C.).

EXAMPLE 2 Crystallization of Calcineurin A/ Calcineurin B/FKBP12/FK506

A binary complex of recombinant bovine FKBP12 and FK506 was prepared,essentially as described previously [K. P. Wilson et al., ActaCrystallography, in press (1995)]. This complex was exchanged intobuffer C (25 mM Tris-HCl, 0.1 mM MnCl₂, 0.1 mM CaCl₂, 2 mMβ-mercaptoethanol, pH 8.0 at 4° C.), and then combined with the purecalcineurin major isoform at a molar ratio of 1:1.3(calcineurin:FKBP/FK506 complex) and a final total protein concentrationof 1-2 mg/ml. The calcineurin/FKBP12/FK506 complex was allowed toequilibrate for 1 hour at 4° C., before the addition of clostripain(IUB:4.4.22.8; Worthington) at 3 mg of clostripain per 100 mg ofcomplex. Proteolytic digestion of the complex was allowed to proceed for3-4 days at 4° C., before the protein was concentrated to 15-20 mg/ml(50-100 mg total protein) by ultrafiltration, and then size-fractionatedat 4° C. on a column system of Sephacryl S-300 HR (2.6×100 cm×3)equilibrated in the same buffer (buffer C). The central 90% of the mainpeak, eluting soon after the void volume, was pooled. The pooledmaterial was analyzed by SDS-polyacrylamide gel electrophoresis,reversed phase HPLC, UV absorption spectroscopy and electrospray massspectroscopy. In addition, direct N-terminal amino acid sequenceanalyses were performed on each of the polypeptide components present,and on HPLC-purified peptides from additional proteolytic mappingexperiments which generated a range of internally truncated peptides.These analyses indicated that pooled material consisted, predominantly,of intact complex, containing (in approximately 1:1:1:1 stoichiometry):

-   -   a) N- and C-terminally truncated calcineurin A (residues        17-392);    -   b) intact, myristoylated calcineurin B (residues 1-169);    -   c) intact FKBP12; and    -   d) FK506.        The added clostripain, unbound FKBP-12/FK506 complex, and the        small peptide fragments (generated by clostripain-proteolysis of        the calcineurin) eluted later from the size-exclusion column and        were discarded.

The proteolytic processing of the complex under these conditions doesnot give single pure products for either the calcineurin A or thecalcineurin B. The product initiation and termination points are allextremely similar, but not identical. For the A chain, cleavage at theN-terminus results in about 95%+ of the products beginning at residue17. Approximately 5% of the products begin at residue 20. The majorityof the C-terminal cleavage leaves products that appear to terminate atresidue 392, since no evidence was found for other C-terminaltermination points. For the calcineurin B, the majority of the productsremain N-terminally blocked and, therefore, are still myristoylated, asdemonstrated by the crystal structure. A small proportion (perhaps up to25%) appears to be cleaved between residues 4 (Ala) and 5 (Ser). Noevidence was found for C-terminal cleavage of the calcineurin B.

The purified complex was concentrated by ultrafiltration to 25-55 mg/ml,and centrifuged at 40,000×g for 10-15 minutes. Crystals were obtainedfrom these solutions, using hanging drops (initially 8 μL total, 25%precipitant) suspended over a precipitant reservoir of 8% PEG 8000, 0.1M potassium phosphate, 20 mM β-mercaptoethanol. Crystals appeared within3-4 days at 4° C., and reached maximal size in about 2-3 weeks. SDS PAGEanalysis of the redissolved crystals showed essentially identicalprotein composition as the original complex solution.

Those of skill in the art will appreciate that the aforesaidcrystallization conditions can be varied. Such variations may be usedalone or in combination, and include final protein/inhibitor complexconcentrations between 5 mg/ml and 35 mg/ml; all combinations ofcalcineurin/FKBP12/FK506 to precipitant ratios; citrate concentrationsbetween 1 mM and 200 mM; DTT concentrations between 0 mM and 10 mM; andany concentration of β-mercaptoethanol; pH ranges between 5.5 and 9.5;PEG concentrations between 10% and 25% (g/100 ml); PEG weights between2000 and 8000; LiSO₄ concentrations between 50 and 750 mM; HEPESconcentrations between 5 and 395 mM; and any concentration or type ofdetergent; any temperature between −5° C. and 30° C.; andcrystallization of calcineurin/FKBP12/FK506 complexes by batch, liquidbridge, or dialysis method using these conditions or variations thereof.

EXAMPLE 3 Crystal Structure of Calcineurin A/ Calcineurin B/FKBP12/FK506

Initial heavy atom searches were carried out with crystals stabilized in50 mM HEPES, pH 7.5, and 12% PEG 8000. Native and heavy atom derivatizedcrystals were transferred to 50 mM HEPES, pH 7.5, 12% PEG 8000, and 22%glycerol (w/v), and frozen at approximately −165° C. in a dry nitrogengas stream for data collection. This stabilization process changed theunit cell dimensions to a=89.3 Å, b=92.1 Å, and c=118.5 Å. Twoderivatives were obtained under these conditions usingdi-Q-iodobis(ethylenediamine)-di-platinum (II) nitrate (PIP), andPb(NO₃)₂, the latter on crystals which had been treated with EGTA toremove Ca⁺⁺ from the metal binding sites on calcineurin B. Native andderivative data sets were collected on frozen crystals by oscillationphotography on a Rigaku R-AXIS IIC phosphor imaging area detectormounted on a Rigaku RU200 rotating anode generator (Molecular StructureCorp., Houston, Tex.), operating at 50 kV and 100 mA. Measuredintensities were integrated, scaled, and merged using the HKL softwarepackage (Z. Otwinowski and W. Minor, personal communication). The nativedata set was denoted native1 and the two derivative data sets denotedcalc_(—)106 (PIP) and calc_(—)149 (Pb(NO₃)₂).

Heavy atom positions were located by inspection or with RSPS (Knight, S.Thesis, Swedish Unv. Agricultural Sciences (1989)) and confirmed withdifference Fourier syntheses using PHASES (W. Furey et al., S. Am.Cryst. Assoc. Mtg. Summ. 18, p. 73 (1990)). Heavy atom parameters wererefined with HEAVY (T. C. Terwilliger et al., Acta Crystalloqr., A39,pp. 813-817 (1983)), and phases computed using either HEAVY or MLPHARE(Z. Otwinowski, ML-PHARE CCP4 Proc. 80-88 (Daresbury Laboratory,Warrington, UK, 1991)). MIR phases were improved and extended by cyclesof solvent flattening (B. C. Wang, Meth. Enzym. 115, pp. 90-112 (1985)),phase combination using SIGMAA (R. J. Reed, Acta Crystallogr., A42, pp.140-149 (1986), and histogram matching combined with Sayre's equation(K. Y. J. Zhang et al., Acta Crystallogr., A46, pp. 377-381 (1990) usingthe CCP4 crystallographic package (CCP4 (1986), A Suite of Programs forProtein Crystallography, SERC Daresbury Laboratory, Warrington WA4 4WD,England). The molecular model was built into electron density maps usingQUANTA (Quanta version 4.1, Molecular Simulations Inc., BurlingtonMass., 1995), and the model refined with XPLOR-3.1 (A. T. Brunger,X-PLOR (Version 3.1), Yale Univ., New Haven, (1993)).

Refinement of the PIP and Pb(NO₃)₂ derivatives, including the anomalouscontribution from the Pb(NO₃)₂ derivative gave a figure of merit of 0.53to 4.6 Å. The resulting MIRAS map was subjected to cycles of solventflattening, phase combination, and phase extension to produce anelectron density map at 4.0 Å. A partial model for calcineurin A andcalcineurin B was built into this map, and FKBP12 was positioned intothe density as a rigid body. The partial structure was refined againstthe native1 data, and then refined as a rigid body against a new nativedata set (denoted native2), from crystals stabilized in 0.1M potassiumphosphate, pH 7.5, 12% PEG 8000, and cryoprotected with O.1M potassiumphosphate, pH 7.5, 9% PEG 8000, and 23% ethylene glycol. Theseconditions yielded yet another cell, with dimensions a=91.3 Å, b=94.4 Å,and c=116.8. Three new derivatives soaked and cryoprotected under thesame conditions as native2 were obtained with HgCl₂ (calc_(—)158),Pb(NO₃)₂ (calc_(—)171), and K₂PtCl₄ (calc_(—)170). Heavy atom parameterrefinement for these three derivatives against native2 to 3.3 Å gave afinal figure of merit of 0.58, including the Pb(NO₃)₂ anomalous data.This MIRAS map was again subjected to cycles of solvent flattening/phaseextension, and multiple electron density maps were calculated rangingfrom 3.6 to 2.6 Å resolution. The resulting maps were used to build inapproximately 80% of CnA and CnB chains as polyalanine, beginning withthe previous model. Multiple rounds of model building, positionalrefinement, phase combination, and phase extension gave improvedelectron density maps into which a nearly complete model was built. Thepositions of the lead, mercury, and platinum heavy atom sites were usedto confirm the register of the sequence during building of loops andside chains in the latter stages of model building. A nearly completemodel of the CnA/CnB/FKBP12/FK506 complex was subjected to simulatedannealing refinement, followed by positional and temperature factorrefinement at 2.6 Å. The remainder of the model along with well orderedwater molecules was built into 2|F_(o)|−F_(c)| and |F_(o)|−|F_(c)|difference fourier maps. The current model contains residues 24-240 and247-370 of calcineurin A, residues 5-82 and 84-168 of calcineurin B, anN-terminal myristoyl group associated with calcineurin B, residues 1-107of FKBP12, FK506, 4 Ca⁺⁺ ions in the calcineurin B Ca⁺⁺ binding sites, 1PO₄ ⁻⁻ group, 1 Fe⁺⁺ ion, and 1 Zn⁺⁺ ion in the calcineurin A activesite, and 87 waters. It has been refined using data between 6.0 and 2.5Å.

EXAMPLE 4 Structural Features Of The CnA/CnB/FKBP12/FK506 Crystal

The crystals had an orthorhombic space group symmetry P₁₂₁₂₁. Thecrystals also had a rectangular shaped unit cell, each unit cell havingthe dimensions a=90±5 Å, b=94±6 Å, and c=117±5 Å. The crystal comprisedfour complexes per unit cell, wherein CnA interacts with CnB, FKBP12,and FK506; CnB interacts with CnA, FKBP12, and FK506; FKBP12 interactswith CnA, CnB, and FK506; and FK506 interacts with CnA, CnB, and FKBP12.

The CnA subunit contained a series of amino acids within 8 Å of aphosphate group and two metal ions bound to the active site. These wereamino acids 90, 91, 92, 118, 120, 121, 122, 150, 151, 156, 160, 199,232, 253, 254, 256, 281, 282, 283, 284, 306, 311, 312, and 317,according to FIG. 1. A subset of these, amino acids 90, 91, 92, 118,120, 121, 122, 150, 151, 156, 160, 281, 282, 283, 306, 311, 199, 232,and 254, where within 5 Å.

The crystal further contained a FKBP12/FK506 binding site made up of CnAamino acids 122, 124, 159, 160, 310, 312, 313, 314, 339, 341, 343, 344,345, 347, 351, 352, 353, 354, 355, 356, 359, 360, and 363 and CnB aminoacids 49, 50, 114, 115, 118, 119, 121, 122, 123, 124, 157, 158, 159,161, and 162.

The components of the quarternary complex that make up the crystalassociated to form a roughly rectangular structure with overalldimensions of 87×61×37 Å. CnA was the largest component of the complexand consisted mainly of a globular domain which contains the phosphatasesite. This phosphatase-containing domain was characterized by aβ-sandwich motif which formed the core of the enzyme. Perhaps the moststriking feature of the quarternary complex was a 22 residue α-helixwhich extended nearly 40 Å away from the surface of thephosphatase-containing domain and contained the CnB binding helix (BBH).CnB comprised two calmodulin-like domains that, taken together, formed ahydrophobic groove which interacted with the upper surface of thecalcineurin binding helix, leaving the underside completely exposed. Itis to this exposed region of the BBH that the FKBP12-FK506 complexbound.

Architecture of Calcineurin A

The CnA fragment used in this study contained only the phosphatasedomain and the CnB binding region (residues 17-392). Missing from thisfragment were the calmodulin binding domain and the autoinhibitoryregions. The phosphatase domain formed a compact α/β sandwich while theCnB binding region consisted of a short linker followed by a singleα-helix that protruded from the phosphatase domain.

Phosphatase Domain

The phosphatase domain of CnA formed an ellipsoid with approximatedimensions 35 Å×35 Å×45 Å. The core of the domain consisted of two mixedβ-sheets, termed sheet 1 and sheet 2, which were flanked on one side bya mixed α/β structure and on the other side by an all α structure. Thetwo central β-sheets formed a distorted β-sandwich which contains anopen and closed end. At the closed end of the β-sandwich sheet 2extended above sheet 1, giving the β-sandwich an overall appearancesimilar to the greek letter λ. The two sheets formed an angle ofapproximately 30°, resulting in their gradual separation from closed toopen end. The inner core of the β-sandwich was filled almost exclusivelywith hydrophobic residues, with smaller side chains residing at theclosed end of the core and larger, more bulky side chains filling theopen end. Strands β6, β10, and β12 from sheet 1 were parallel and ran inthe direction of the closed end of the sandwich, as did strands β4, β3,β2, and β14 in sheet 2. Following β14, the sequence formed an extendedregion covering approximately 24 residues before the start of the BBH.Residues 340-348, while still part of the phosphatase core, participatedin multiple contacts with CnB and thus can be considered as part of theCnB binding region. Additional contacts between CnA and CnB occurred atthe N-terminus of CnA and in loop L1 where a salt bridge is formedbetween Glu-53 of CnA and Lys-134 of CnB. These interactions appeared tohelp stabilize the extended CnB/BBH structure.

The phosphatase active site was located above the closed end of theβ-sandwich, formed by the convergence of several loops and by a portionof sheet 2 which extends above the β-sandwich. Residues that formed partof the active site were located in loops L2, L3, L4, and L6 and at theC-termini of strands β2 and β3. The somewhat shallow active site pocketwas located in the middle of a larger, curved channel that runs alongthe top of sheet 1 and helix α9. This channel should accommodate accessto the active site by larger, phosphorylated substrates and may helpprovide specificity through interactions with residues surrounding thephosphorylated side chain of the substrate.

The active site contained two metal ion sites that are modelled as Zn²⁺and Fe³⁺, as well as a single phosphate ion, whose presence in thestructure may be the result of including 100 mM potassium phosphatebuffer in the crystallization conditions.

The Zn²⁺ and Fe³⁺ atoms were separated by approximately 3.0 Å in CnA andwere identified in the active site on the basis of their interactionswith surrounding ligands. The zinc was coordinated by the side chainsAsp-118(Oλ2), Asn-150(Oλ1), His-199(Nε2) and His-281(Nε1), and by aphosphate oxygen. The iron was coordinated by Asp-90(Oλ1), His-92(Nε2),Asp-118(Oλ2), a phosphate oxygen, and a water molecule.

The bound phosphate, in addition to coordinating both metals, wasstabilized by interactions with the guanidinium groups of Arg-122 andArg-254, and with the Nε2 of His-151. Arg-254 extended down from loopL5, which is fully 8.5 Å away from the phosphate group, and wasstabilized through a bidentate interaction with the carboxylate ofAsp-234. His-151 was situated in the active site within hydrogen bondingdistance of the most solvent exposed phosphate oxygen. Its side chainposition was stabilized by a hydrogen bond to Asp-121, while the mainchain conformation was stabilized by the next residue, Glu-152, whichmakes a pair of hydrogen bonds to main chain nitrogens surrounding themetal-bridging ligand, Asp-118.

Calcineurin B Binding Helix

The BBH was a five turn amphipathic α-helix (residues 350-370) to whichboth CnB and the binary FKBP12-FK506 complex bound. The top half of theBBH was completely non-polar and formed a complementary surface to thehydrophobic groove formed by CnB (see below). The tip of the BBH abuttedthe N-terminal helix of CnB, which lay perpendicular to the axis of BBHand caps the end of the BBH binding groove. The lower half of the BBHhelix was polar except for a small hydrophobic patch near itsN-terminus. This patch formed part of the contact surface with theFKBP12-FK506 complex.

Architecture of Calcineurin B

The structure of CnB consisted of two globular calcium-binding domainsflanked by a long C-terminal β-strand. Each calcium-binding domaincontained two Ca²⁺-binding EF-hand motifs. Domain 1 (residues 1 to 84)connected to domain 2 (residues 86 to 155) via an α-helix that waskinked at Gly-85. Domains 1 and 2 were arranged linearly along the BBHand, together with the amphipathic C-terminal strand, forms a 33 Å longhydrophobic groove into which the top half of the BBH was embedded.

The three-dimensional structure of each of the pairs of EF-hands (EF1and EF2 in domain 1, and EF3 and EF4 in domain 2) in CnB was highlyconserved with those of other members of the super-family; theintra-domain calcium-calcium distances in CnB were nearly identical tothose found in calmodulin, for example. In all four EF hands the Ca²⁺ion was coordinated by five ligands. These are Asp-30, Asp-32, Ser-34,Glu-41, and the Ser-36 carbonyl oxygen for EF1, Asp-62, Asp-64, Asn-66,Glu-73, and the Gly-68 carbonyl oxygen for EF2, Asp-99, Asp-101,Asp-103, Glu-110 and the Tyr-105 carbonyl oxygen for EF3, and Asp-140,Asp-142, Asp-144, Glu-151 and the Arg-146 carbonyl oxygen for EF4.

The N-terminal glycine of CnB was covalently linked to myristate, a 14carbon saturated fatty acid. The myristate group was located at theextreme end of the calcineurin complex, near the end of the BBH (FIG.2). It was connected to the N-terminal helix by a 15 residue loop, andlay against and ran parallel to the N-terminal helix of CnB, which wasitself hydrophobic.

Structure of FKBP12-FK506

The conformation of FKBP12 in the ternary complex was nearly identicalto that found in the structure of the FKBP12-FK506 binary complex (vanDuyne et al., 1991a; Becker et al., 1993; Wilson et al., in press).Superposition of FKBP12 from the ternary and binary complexes gave aroot-mean-square (rms) deviation of 0.59 Å for Cα atoms. Similarly, theconformations of FK506 were almost identical in the two complexes, withan rms difference of 0.21 Å for all non-hydrogen atoms, excluding thosefrom the highly flexible C21 allyl group. However, the relative positionof FK506 to FKBP12 differed in the ternary and binary complexes. In theternary complex FK506 was rotated by about 80 from the body of FKBP12,resulting in a displacement of 1.7 Å for the C21 carbon at the base ofthe allyl group. A concomitant displacement of the His-87 to Ile-90 loopin FKBP12 was observed as well. This rotation allowed the allyl, and toa lesser extent, cyclohexyl moieties of FK506 to more intimately contactthe BBH. One consequence of this rotation was the loss of a hydrogenbond between the Glu-54 carbonyl oxygen of FKBP12 and the C24 hydroxylgroup of FK506 in the ternary complex.

FKBP12-FK506 Binding to Calcineurin

The FKBP12-FK506 complex bound to calcineurin at the base of the BBHmaking contacts with the BBH, CnB, and the phosphatase domain of CnA.The solvent accessible surface area lost to each component uponFKBP12-FK506 binding was 320 Å², 479 Å², and 512 Å². TheFKBP12-calcineurin contacts surrounded the FK506 ligand and clustered tothree distinct regions of the FKBP12 sequence: His-87 to Ile-90, Asp-37to Asp-41, and Arg-42 to Phe-46. These regions contacted the BBH, thephosphatase domain, and CnB, respectively.

The principal site of interaction between FK506 and calcineurin was apredominantly hydrophobic cleft located at the interface of CnB and theBBH. Side chains that formed the cleft came from residues Leu-343,Pro-344, Trp-352, Ser-353 (Cβ), and Phe-356 on the BBH and residuesLeu-115, Met-118, Val-119 and Leu-123 from CnB. This binding cleft wasapproximately 8 Å long, and had surface properties complementary to theC15-C21 region of FK506. The majority of contacts made by FK506 werefrom C15 through C17 and the C21 allyl group. The allyl group extendedinto a deep pocket within the hydrophobic cleft, making a number offavorable van der Waals contacts with main chain and side chain atoms.The FK506-calcineurin interaction was further stabilized by an unusualbifurcated hydrogen bond between Nε1 of Trp-352 and the C13 and C15methoxy oxygens of FK506.

FIG. 2 depicts the structure of the calcineurin A, calcineurin B,FKBP12, and FK506 subunits as determined by x-ray crystallography.

EXAMPLE 5 Use of Calcineurin A/Calcineurin B/FKBP12/FK506

The coordinates in FIG. 1 are used to design compounds, includinginhibitory compounds, that associate with calcineurin or homologues ofcalcineurin, directly or through prior complexation with FKBP12 or aFKBP12 homologue. This process may be aided by using a machine-readabledata storage medium encoded with a set of machine-executableinstructions, wherein the recorded instructions are capable ofdisplaying a three-dimensional representation of theCnA/CnB/FKBP12/FK506 complex or a portion thereof. The graphicalrepresentation is used according to the methods described herein todesign compounds, including an inhibitory compound, that bind tocalcineurin. Such compounds may associate with calcineurin at the activesite, the FKBP12/FK506 binding site, or both sites or at adjacent areato either or both of these sites.

FKBP12/FK506 Binding Site Inhibitors

The process outlined above is used to design a compound that inhibitcalcineurin by associating with the FKBP12/FK506 binding site. Such acompound binds first to FKBP12 or a variant of FKBP12 and thenassociates with the FKBP12/FK506 binding site. This compound consists ofa hydrophobic moiety capable of making van der Waal's contact with oneor more of the following residues on FKBP12: Trp59, Phe46, Tyr26, Val55,Phe99, and Ile56. Substituted onto this hydrophobic core is a hydrogenbond acceptor capable of forming a hydrogen bond with Ile56 on FKBP12and a second hydrogen bond acceptor or a hydrogen bond donor that canform a hydrogen bond with Tyr82 on FKBP12. The binding core alsocontains a linker that projects a hydrophobic moiety 10-14 Å from thecenter of the binding core to make van der Waal's contact with one ormore of the following residues: Leu115, Val119, Met118 of CnB andTrp352, Ser353, Phe356 and Val357 on CnA. The binding core also containsa second linker that projects a moiety 7-11 Å from the center of thebinding core to make van der Waal's contact with one or more of thefollowing residues: Leu123 on CnB and Leu343, Tyr341, Pro344 and Trp352on CnA and may optionally act as a hydrogen bond acceptor with Trp352and/or a hydrogen bond donor or acceptor with Tyr341. The binding corecontains a third linker that projects a moiety 8-12 Å from the center ofthe binding core to make van der Waal's contact with one or more of thefollowing residues: Pro355 and Phe356 and may optionally act as ahydrogen bond donor with Glu359 on CnA. This third linker will also makevan der Waal contact with Tyr82, Ile56 and His87 on FKBP12. Thismolecule contains fewer than three secondary amide bonds and has amolecular weight of less than 1000.

Active Site Inhibitors

In another example, the process outlined above is used to designcompounds that inhibit calcineurin by associating directly with thephosphatase active site. Such compounds will contain either a phosphateresidue or a surrogate for a phosphate residue and additionalfunctionality that imparts affinity for calcineurin.

The process outlined above could also be used to design compounds thatinhibit calcineurin by blocking access to the active site. Examples ofclefts in the enzyme that may be blocked are described by the followinggroups of CnA amino acids, as set forth in FIG. 1:

a) Arg122, Phe125, Asp313, His339, Pro340, Tyr341, Trp342, Phe346,Tyr124, Thr161, Pro344, Phe160 and Asn345;

b) Trp232, Leu231, Pro221, His155, Glu220, Leu156, Cys153, Asn150,Pro222, Cys256, Gly255 and Arg148; and

c) Leu302, Pro235, Glu282, Phe239, Tyr291, Gln284, Thr304, Phe259,Leu236, Glu237, Val253, Ala283 and Asp234.

Compounds that make contact primarily with any of these three sets ofresidues would be active site inhibitors.

EXAMPLE 6 Calcineurin Inhibition Assay

The calcineurin assay is performed essentially as described by Klee andCohen [C. V. Klee et al., Moli. Aspects Cell. Regul., 5, pp. 225-248(1988)].

A commercial preparation of bovine brain calcineurin is used (Sigma,Cat# C-1907, specific activity=16 nmol/min/mg under the conditions ofthe assay). Radiolabeled phosphorylated peptide substrate, derived fromthe serine phosphorylation site sequence of the RII subunit ofcAMP-dependent protein kinase, is prepared as described previously [R.A. Aldape et al., J. Biol. Chem. 267, pp. 16029-16032 (1992)].

The serine phosphatase assay is performed in 60 μl buffer containing 20mM Tris, pH 8.0, 0.1M NaCl, 6 mM MgCl₂, 0.1 mM CaCl₂, 0.5 mMdithiothreitol, and 0.1 mg/ml bovine serum albumin (C. V. Klee et al,supra). The following ordered additions are made for the assays: 5 nM-15μM FKBP, 5 nM-15 μM FK506, 160 nM bovine calmodulin (Sigma, Cat# P-277)and 40 nM bovine brain calcineurin. [³²P]-phosphorylated peptide isadded to 1-2 μM final concentration, followed by a 15 min incubation at30° C. Reactions are quenched with 540 μl 0.1M potassium phosphate/5%trichloracetic acid (w/v). Cation exchange columns (Dowex AG1-X8, 0.6ml) are used for separation of free [³²P]-P_(i) (M. J. Hubbard et al.,in Molecular Neurobiology: A Practical Approach, J. Chad et al., Eds.(Oxford University Press, Oxford, England) pp. 135-149, 1991). Thequenched reaction mixtures (0.6 ml) are applied to the columns, followedby a 0.6 ml H₂O wash, and the effluents are collected in scintillationvials and counted with 5 ml of scintillation cocktail (BeckmannLiquiscint). All assays are performed in duplicate.

Affinity of the FKBP-test ligand complexes for calcineurin is determinedby varying the concentrations of FKBP and test ligand at 30° C., using adrug:FKBP ratio of 1.35:1. FK506:test ligand ratios are increasedappropriately for the lower affinity ligands to ensure saturation of theFKBP with test ligand.

Data Analysis

The inhibition constant for calcineurin by the FKBP/test ligandcomplexes (K_(ic)) is calculated by computer-fitting the fractionalinhibition data as a function of concentration of free FKBP and testligand to an the equilibrium equation derived by Liu et al [J. Liu, etal., Biochemistry, 31, pp. 3896-3901 (1992)]. Quadratic equations arefirst used to calculate the free concentrations of these reactioncomponents from the concentrations of calcineurin, FKBP and test ligandin the experiment, as well as the K_(i) of the FKBP for test ligand. Thecalcineurin affinity of the FKBP/test ligand complex is calculated usingthe equation: I/(1−I)=[TestLigand]_(free)[FKBP12]_(free)/(K_(i)K_(ic)),where I is the fractional inhibition of calcineurin and (1-I) is thefractional activity remaining. K_(ic) and the associated standarddeviation are calculated from the linear regressions performed onMiniTab (Addison-Wesley).

EXAMPLE 7 Immunosuppression (Mitogenesis) Assays Cell Source and Culture

Fresh peripheral blood lymphocytes (PBLs) from LeukoPak cells or wholeblood from random normal blood donors (tested HIV-negative and hepatitisnegative) are isolated and separated by density centrifugation overHistopaque 1077 (Sigma Chemical Co., St. Louis, Mo.). The murine CTLLcytotoxic T cell line and the human Jurkat T cell line are availablefrom ATCC (CTLL-2 ATCC TIB214, JURKAT CLONE E6-1 ATCC TIB152). The humanallogeneic B cell lines used for activation of the fresh PBLs areEBV-transformed lymphocytes from normal healthy adult donors with twocompletely different HLA haplotypes. All cell lines are routinely testedfor the presence of Mycloplasma contamination using the Gibco Mycotecttest kit and found to be Mycoplasma-free. Culture medium consisted ofRPMI 1640 (Gibco, Grand Island, N.Y.) containing penicillin (50 U/ml)and streptomycin (50 μg/ml), L-glutamine 2 mM, 2 mercaptoethanol(5×10⁻⁵), 10% heat-inactivated FCS and 10 mM HEPES.

Compound Solutions and Titrations

All chemical stocks are dissolved in DMSO. Titrations of compounds aremade into the medium the individual assay are carried out in, i.e.,complete RPMI or HB 104 for final diluted concentrations, using multiplethree-fold dilutions from 1 μM or 10 μM stock solutions.

Mitocenesis Assays (“PMA” and “OKT3”)

The inhibitory effect of test compounds on the proliferation of humanPBLs in response to mitogens (W. K. Waithe et al., Handbook ofExperimental Immunology, 3d Ed., Blackwell Scientific Publications,Oxford (1978); B. B. Mishell et al., Selected Methods in CellularImmunology, W.H. Freeman and Co., San Francisco, Calif. (1980)) areassessed by stimulation of 5×10⁴ cells with OKT3 (10⁻⁴ dilution final)or PMA (10 ng/ml) plus ionomycin (250 ng/ml) in the presence or absenceof different concentrations of test compounds and control drugs (CsA,FK506, rapamycin) in final volume of 200 μl per well in 96 well roundbottomed plates. After 48 h incubation (37° C., 5% CO₂), cells arepulsed with 1 μCi of ³H-Leucine, harvested 24 h later with a Tom Tekcell harvester, and counted in LKB β-scintillation counter. Results(cpm) are compared with controls with medium alone, and concentrationscausing 50% reduction in counts (IC₅₀) are calculated.

While we have described a number of embodiments of this invention, it isapparent that our basic examples may be altered to provide otherembodiments which utilize the products and processes of this invention.Therefore, it will be appreciated that the scope of this invention is tobe defined by the appended claims rather than by the specificembodiments which have been represented by way of example.

1. A crystallized molecule or molecular complex comprising a bindingpocket defined by structure coordinates of CnA amino acids 90, 91, 92,118, 120, 121, 122, 150, 151, 156, 160, 199, 232, 253, 254, 256, 281,282, 283, 284, 306, 311, 312, and 317 according to FIG. 1, or ahomologue of said molecule or molecular complex wherein said homologuecomprises a binding pocket that has a root mean square deviation fromthe backbone atoms of said amino acids of not more than 1.5 Å.
 2. Thecrystallized molecule or molecular complex according to claim 1, whereinsaid binding pocket is defined by structure coordinates of CnA aminoacids 90, 91, 92, 118, 120, 121, 122, 150, 151, 156, 160, 199, 281, 282,283, 306, 311, 232, and 254, according to FIG. 1, or a homologue of saidmolecule or molecular complex, wherein said homologue comprises abinding pocket that has a root mean square deviation from the backboneatoms of said amino acids of not more than 1.5 Å.
 3. A crystallizedmolecule or molecular complex comprising a binding pocket defined bystructure coordinates of CnA amino acids 122, 124, 159, 160, 310, 312,313, 314, 339, 341, 343, 344, 345, 347, 351, 352, 353, 354, 355, 356,359, 360, and 363; and CnB amino acids 49, 50, 114, 115, 118, 119, 121,122, 123, 123, 157, 158, 159, 161, and 162 according to FIG. 1, or ahomologue of said molecule or molecular complex, wherein said homologuecomprises a binding pocket that has a root mean square deviation fromthe backbone atoms of said amino acids of not more than 1.5 Å.
 4. Thecrystallized molecule or molecular complex according to claim 1, furthercomprising a second binding pocket defined by CnA amino acids 122, 124,159, 160, 310, 312, 313, 314, 339, 341, 343, 344, 345, 347, 351, 352,353, 354, 355, 356, 359, 360, and 363; and CnB amino acids 49, 50, 114,115, 118, 119, 121, 122, 123, 124, 157, 158, 159, 161, and 162;according to FIG. 1, or a homologue of said molecule or molecularcomplex, wherein said homologue comprises a second binding pocket thathas a root mean square deviation from the backbone atoms of said aminoacids of not more than 1.5 Å.
 5. The crystallized molecule or molecularcomplex according to claim 4, wherein said molecule or molecular complexis defined by the set of structure coordinates according to FIG. 1, or ahomologue thereof, wherein said homologue has a root mean squaredeviation from the backbone atoms of said amino acids of not more than1.5 Å.
 6. The crystallized molecule or molecular complex according toclaim 4, wherein said molecule or molecular complex comprises aminoacids 17-392 of CnA, CnB, FKBP12 and FK506. 7-15. (canceled)
 16. Amethod for preparing a CnA/CnB/FKBP12/FK506 crystal comprising the stepsof: a. forming a molecular complex between FKBP12, FK506, calcineurin Aand calcineurin B, wherein the calcineurin A lacks a calmodulin bindingdomain and an autoinhibitory domain; and b. crystallizing the digestedcomplex.
 17. The method according to claim 16, wherein the calmodulinbinding domain and the autoinhibitory domain of said calcineurin A areremoved by proteolytic digestion with a protease selected fromclostripain, trypsin, endoproteinase Lys-C, endoproteinase Asp-N,endoproteinase Glu-C, elastase, enterokinase, restriction proteaseFactor Xa, thermolysin, Il-1 beta converting enzyme or HIV-1 protease.18. The method according to claim 17, wherein the protease isclostripain and the calcineurin A subunit in the crystallized complexhas a molecular weight of about 42 kDa.